The broad objective of this study is to identify molecular targets regulating zebrafish craniofacial skeletogenesis and primary and replacement tooth formation (RTF), that can be manipulated to repair human craniofacial skeletal and tooth defects in humans. Although significant research efforts have resulted in the identificaton of certain gene mutations responsible for human craniofacial skeletal defects, functional characterizations have been hampered by limited availability of human tissues and suitable animal models. Similarly, clinically relevant biological therapies to regenerate teeth do not currently exist, and research on tooth regeneration strategies is hampered by similar limitations. Targeted, molecular based therapies to effectively and permanently correct mineralized craniofacial and tooth defects would be a significant advancement over current surgical repair methods. The fact that zebrafish craniofacial skeletal development closely resembles that of mammals, combined with the fact that zebrafish continuously regenerate teeth throughout their lives, provides a unique opportunity to define and funcitionally characterize molecular signaling cascades directing mineralized craniofacial skeletal and replacement tooth development. The extensive conservation of gene identity, genomic organization and gene function between zebrafish and humans allows for studies in zebrafish to be directly related to human development. Our long term goal is to identify genetic pathways that can be manipulated to repair craniofacial skeletal defects and initiate RTF in humans. The objective of this study is to exploit the zebrafish to perform functional studies of molecular mechanisms regulating craniofacial mineralized skeletal and tooth formation. In this proposal we will first perform a forward genetic ENU mutagenesis screen, combined with a sensitive in vivo assay for mineralized tissue formation, to identify novel craniofacial skeletal and RTF mutants. Next, we will confirm the molecular identity, and perform functional characterization, of each identified mutant. We anticipate that the proposed studies will reveal molecular signaling cascades that may facilitate the implementation of clinically relevant gene based therapies to correct mineralized craniofacial skeletal and tooth defects in humans. [unreadable] [unreadable] [unreadable]